Cellular Uptake Of Propidium Iodide Research Articles

Transitory phases of autophagic death and programmed necrosis during superoxide-induced neuronal cell death

Neurons can undergo a diverse range of death responses under oxidative stress, encompassing apoptosis (caspase-dependent, programmed cell death) to various forms of caspase-independent death, including necrosis. We recently showed that primary murine cortical neurons exposed acutely to hydrogen peroxide undergo caspase-independent death, both autophagic cell death and programmed necrosis. To determine how oxidative stress induced by superoxide affects the route to cellular demise, we exposed primary cortical neurons to extended superoxide insult (provided by exogenous xanthine and xanthine oxidase in the presence of catalase). Under these conditions, over 24h, the nitroblue tetrazolium-reducing activity (indicative of superoxide) rose significantly during the first 4 to 8h and then declined to background levels. As with hydrogen peroxide, this superoxide insult failed to activate downstream caspases (−3, −7, and −9). Substantial depolarization of mitochondria occurred after 1h, and nuclear morphology changes characteristic of oxidative stress became maximal after 2h. However, death indicated by plasma membrane permeabilization (cellular uptake of propidium iodide) approached maximal levels only after 4h, at which time substantial redistribution to the cytosol of death-associated mitochondrial intermembrane space proteins, notably endonuclease G, had occurred. Applying established criteria for autophagic death (knockdown of Atg7) or programmed necrosis (knockdown of endonuclease G), cells treated with the relevant siRNA showed significant blockade of each type of cell death, 4h after onset of the superoxide flux. Yet at later times, siRNA-mediated knockdown failed to prevent death, monitored by cellular uptake of propidium iodide. We conclude that superoxide initially invokes a diverse programmed caspase-independent death response, involving transient manifestation in parallel of autophagic death and programmed necrosis. Ultimately most neurons become overwhelmed by the consequences of severe oxidative stress and die. This study reveals the multiple phases of neuronal cell death modalities under extended oxidative stress.

Immunotoxic depletion of microglia in mouse hippocampal slice cultures enhances ischemia-like neurodegeneration

Microglial cells have been attributed both neuroprotective and neurodegenerative roles in cerebral ischemia. This study presents an immunotoxic method for depletion of microglia from mouse hippocampal slice cultures and the effect of this on ischemia-like oxygen–glucose deprivation (OGD). For microglial depletion hippocampal slice cultures were exposed for 7 days to saporin coupled to an antibody against the microglial receptor Mac1 (Mac1–sap). When subjected to OGD immediately thereafter, resulting neurodegeneration was quantified as cellular uptake of propidium iodide (PI). Cultures were processed 1, 7 or 14 days after OGD for general cell staining and immunohistochemistry for neuronal, astroglial and microglial markers. Following Mac1–sap treatment there was a near total loss of microglia, these microglia-depleted cultures displayed a significant increase in PI uptake and astrogliosis 1 day after OGD compared to non-depleted cultures. In cultures surviving 7 and 14 days after OGD there was a decrease in PI uptake compared to 1 day after OGD. At 7 and 14 days after OGD the differences in Mac1–sap treated or non-treated cultures were still noticeable in terms of more neuron loss in cultures deprived of microglia, while the astroglial reactivity seemed to equalize. Based on the finding that depletion of microglia significantly increased OGD-induced CA1 pyramidal cell degeneration, we conclude that microglia at least in the initial phase of injury exert a neuroprotective role in mouse hippocampal slice cultures.

Thrombin Promotes Release of ATP from Lung Epithelial Cells through Coordinated Activation of Rho- and Ca2+-dependent Signaling Pathways

Extracellular ATP controls key aspects of lung function via activation of epithelial cell purinergic receptors, but how ATP is released from cells remains poorly understood. To identify mechanistic components upstream of ATP release, we examined the effect of selected G protein coupled-receptor activation on ATP release from lung epithelial cells. The protease-activated receptor (PAR) agonist thrombin elicited a rapid Ca(2+)-dependent release of ATP from A549 cells. In contrast, the P2Y(2) receptor agonist UTP caused negligible ATP release, despite promoting a robust Ca(2+) response. Agonist-elicited ATP release was associated with Rho activation and was reduced in cells transfected with dominant negative mutants of p115-Rho GEF or RhoA, and by inhibitors of Rho kinase (ROCK). However, RhoA activation alone did not promote ATP release if temporally separated from Ca(2+) mobilization. PAR3 was the only PAR subtype detected in A549 cells by reverse transcription-PCR. Transfection of cells with human PAR3 cDNA increased thrombin-promoted ATP release, inositol phosphate formation, and RhoA activation. Conversely, small interference RNA against PAR3 diminished thrombin-evoked responses. Thrombin-elicited ATP release was accompanied by an enhanced cellular uptake of propidium iodide in a Ca(2+)- and ROCK-dependent manner and was inhibited by connexin/pannexin hemichannel blockers. Our data suggest that thrombin promotes ATP release from A549 cells via Rho- and Ca(2+)-dependent activation of connexin/pannexin hemichannels. The relevance of these findings is highlighted by the observation that exposure of primary cultures of well differentiated human bronchial epithelial cells to thrombin resulted in robust ATP release, which was inhibited by ROCK inhibitors and by connexin/pannexin hemichannel blockers.

Neuroprotective effects of the anti-inflammatory compound triflusal on ischemia-like neurodegeneration in mouse hippocampal slice cultures occur independent of microglia

Microglial cells, known to play key roles in neuroinflammation, can be immunotoxically eliminated from hippocampal slice cultures by treatment with saporin coupled to the microglial receptor Mac1. Considering microglial cells as a target for anti-inflammatory treatment we studied the effects of microglial depletion on anti-inflammatory treatment of mouse hippocampal slice cultures subjected to ischemia-like neurodegeneration, induced by oxygen-glucose deprivation (OGD). Hippocampal slice cultures, derived from 7-day-old mice and grown for 2 weeks, were divided into 8 groups: (1) control cultures; (2) sham-OGD cultures; (3) OGD cultures; (4) OGD cultures treated with triflusal during OGD; (5) microglia-depleted control cultures; (6) microglia-depleted sham-OGD cultures; (7) microglia-depleted OGD cultures; and (8) microglia-depleted OGD cultures treated with triflusal during OGD. The resulting neurodegeneration was quantified by densitometric measurements of cellular uptake of propidium iodide (PI), with focus on the hippocampal CA1 subfield. Subjection of regular cultures to OGD for 30 min induced a significant increase in PI uptake in the CA1 pyramidal cell layer, compared to control cultures. The presence of 100 μM triflusal during OGD protected against OGD-induced neurodegeneration, and reduced the number of OGD-induced NFkB positive-cells correspondingly. Cultures treated with the Mac1-saporin complex for 7 days displayed an almost total loss of microglial cells. When subjected to OGD after microglial depletion, these cultures displayed a significant increase in OGD-induced PI uptake compared to non-depleted cultures. The presence of triflusal during OGD of these cultures reduced neurodegeneration of the irrespective absence of microglia. In accordance with that, the presence of triflusal during OGD significantly inhibited the increase in the number of reactive microglia and proliferative cells in the CA1 pyramidal and dentate granule cell layers. We conclude that immunotoxic microglia depletion significantly increases the susceptibility of CA1 pyramidal cells to neurodegeneration and that the anti-inflammatory drug triflusal still can exert its neuroprotective role following depletion of microglia.

Interaction between neuropeptide Y (NPY) and brain‐derived neurotrophic factor in NPY‐mediated neuroprotection against excitotoxicity: a role for microglia

The neuroprotective effect of neuropeptide Y (NPY) receptor activation was investigated in organotypic mouse hippocampal slice cultures exposed to the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Exposure of 2-week-old slice cultures, derived from 7-day-old C57BL/6 mice, to 8 microm AMPA, for 24 h, induced degeneration of CA1 and CA3 pyramidal cells, as measured by cellular uptake of propidium iodide (PI). A significant neuroprotection, with a reduction of PI uptake in CA1 and CA3 pyramidal cell layers, was observed after incubation with a Y(2) receptor agonist [NPY(13-36), 300 nm]. This effect was sensitive to the presence of the selective Y(2) receptor antagonist (BIIE0246, 1 microm), but was not affected by addition of TrkB-Fc or by a neutralizing antibody against brain-derived neurotrophic factor (BDNF). Moreover, addition of a Y(1) receptor antagonist (BIBP3226, 1 microm) or a NPY-neutralizing antibody helped to disclose a neuroprotective role of endogenous NPY in CA1 region. Cultures exposed to 8 microm AMPA for 24 h, displayed, as measured by an enzyme-linked immunosorbent assay, a significant increase in BDNF. In such cultures there was an up-regulation of neuronal TrkB immunoreactivity, as well as the presence of BDNF-immunoreactive microglial cells at sites of injury. Thus, an increase of AMPA-receptor mediated neurodegeneration, in the mouse hippocampus, was prevented by neuroprotective pathways activated by NPY receptors (Y(1) and Y(2)), which can be affected by BDNF released by microglia and neurons.

Long-term, Repeated Dose In Vitro Neurotoxicity of the Glutamate Receptor Antagonist L-AP3, Demonstrated in Rat Hippocampal Slice Cultures by Using Continuous Propidium Iodide Incubation

Most in vitro models are only used to assess short-term effects of test compounds. However, as demonstrated here, hippocampal slice cultures can be used for long-term studies. The test compound used was the metabotropic glutamate receptor antagonist, L(+)-2-amino-3-phosphonopropionic acid (L-AP3), which is known to be toxic in vivo after subchronic, but not acute, administration. Degenerative effects were monitored by measuring the cellular uptake of propidium iodide (PI; continuously present in the medium) and lactate dehydrogenase (LDH) leakage, and by using a panel of histological stains. Hippocampal slices, derived from 2-3 day old rats and grown for 3 weeks, were subsequently exposed for the next 3 weeks to 0, 10 or 100microM L-AP3, with PI (2microM) in the culture medium. Exposure to 100microM L-AP3 induced severe toxicity after 4-6 days, shown by massive PI uptake, LDH leakage, changes in MAP2 and GFAP immunostaining, and in Nissl and Timm staining. In contrast, 10microM L-AP3 did not induce detectable neuronal degeneration. Treatment with the NMDA receptor antagonist, MK-801, or the AMPA/KA receptor antagonist NBQX, together with 100microM L-AP3, reduced neurodegeneration down to close to control values. It is concluded that continuous incubation of hippocampal slice cultures with PI is technically feasible for use in studies of inducible neuronal degeneration over time.

The Developmental Expression of Fluorescent Proteins in Organotypic Hippocampal Slice Cultures from Transgenic Mice and its Use in the Determination of Excitotoxic Neurodegeneration

Transgenic mice, expressing fluorescent proteins in neurons and glia, provide new opportunities for real-time microscopic monitoring of degenerative and regenerative structural changes. We have previously validated and compared a number of quantifiable markers for neuronal damage and cell death in organotypic brain slice cultures, such as cellular uptake of propidium iodide (PI), loss of microtubule-associated protein 2 (MAP2), Fluoro-Jade (FJ) cell staining, and the release of cytosolic lactate dehydrogenase (LDH). An important supplement to these markers would be data on corresponding morphological changes, as well as the opportunity to monitor reversible changes or long-term effects in the event of minor damage. As a first step, we present: a) the developmental expression in organotypic hippocampal brain slice cultures of transgenic fluorescent proteins, useful for the visualisation of neuronal subpopulations and astroglial cells; and b) examples of excitotoxic, glutamate receptor-induced degeneration of hippocampal CA1 pyramidal cells, with corresponding astroglial reactivity in such cultures. The slice cultures were set up according to standard techniques, by using one-week old pups from four transgenic mouse strains which express fluorescent proteins in their neurons and/or astroglial cells. From the time of explantation, and subsequently for up to nine weeks in culture, the transgenic neuronal fluorescence displayed the expected characteristics of a developmental, in vivo-like increase, including both the number and localisation of cells, as well as the intensity of fluorescence. At that stage and later, the transgenic fluorescence clearly permitted the visualisation of cell bodies, larger and smaller dendritic branches, spines and axons. In separate experiments, with a 24-hour exposure of matured sliced cultures to 100 microM of the glutamate agonist, N-methyl-D-aspartate (NMDA), we observed, by time-lapse recording, a gradual, but rapid loss of fluorescent CA1 pyramidal cells, accompanied by astrogliosis of transgene fluorescent astroglial cells. Based on these results, we consider that organotypic brain slice cultures from transgenic mice, with fluorescent neurons and glia, combined with detailed visualisation by time-lapse fluorescence microscopy, have great potential for investigating both major irreversible and minor reversible structural changes in neurons and glia, induced by neurotoxins and other neurodegenerative compounds and conditions.

Comparison of neuroprotective effects of erythropoietin (EPO) and carbamylerythropoietin (CEPO) against ischemia-like oxygen–glucose deprivation (OGD) and NMDA excitotoxicity in mouse hippocampal slice cultures

In addition to its well-known hematopoietic effects, erythropoietin (EPO) also has neuroprotective properties. However, hematopoietic side effects are unwanted for neuroprotection, underlining the need for EPO-like compounds with selective neuroprotective actions. One such compound, devoid of hematopoietic bioactivity, is the chemically modified, EPO-derivative carbamylerythropoietin (CEPO). For comparison of the neuroprotective effects of CEPO and EPO, we subjected organotypic hippocampal slice cultures to oxygen–glucose deprivation (OGD) or N-methyl- d-aspartate (NMDA) excitotoxicity. Hippocampal slice cultures were pretreated for 24 h with 100 IU/ml EPO (= 26 nM) or 26 nM CEPO before OGD or NMDA lesioning. Exposure to EPO and CEPO continued during OGD and for the next 24 h until histology, as well as during the 24 h exposure to NMDA. Neuronal cell death was quantified by cellular uptake of propidium iodide (PI), recorded before the start of OGD and NMDA exposure and 24 h after. In cultures exposed to OGD or NMDA, CEPO reduced PI uptake by 49 ± 3 or 35 ± 8%, respectively, compared to lesion-only controls. EPO reduced PI uptake by 33 ± 5 and 15 ± 8%, respectively, in the OGD and NMDA exposed cultures. To elucidate a possible mechanism involved in EPO and CEPO neuroprotection against OGD, the integrity of α-II-spectrin cytoskeletal protein was studied. Both EPO and CEPO significantly reduced formation of spectrin cleavage products in the OGD model. We conclude that CEPO is at least as efficient neuroprotectant as EPO when excitotoxicity is modeled in mouse hippocampal slice cultures.

Vasoactive Intestinal Peptide and PACAP38 Control N-Methyl-D-aspartic Acid-induced Dendrite Motility by Modifying the Activities of Rho GTPases and Phosphatidylinositol 3-Kinases

Dendrite morphogenesis is highly dynamic and characterized by the addition and elongation of processes and also by their selective maintenance, retraction, and elimination. Glutamate can influence these events via N-methyl-d-aspartic acid (NMDA) receptors. The neuropeptides vasoactive intestinal peptide and pituitary adenylyl cyclase-activating polypeptide-38 (PACAP38) affect neurogenesis and differentiation in the developing nervous system. We report here that the peptides and NMDA acted synergistically on dendrite and branch formation. In stage III hippocampal neurons, NMDA increased not only the addition but also the elimination of new dendrites and branches by activating Rac and Cdc42 and phosphatidylinositol 3-kinases, respectively. When applied alone, the neuropeptides did not influence dendrite or branch formation. However, they reduced the elimination of newly formed dendrites and branches caused by NMDA by preventing the NMDA-induced activation of phosphatidylinositol 3-kinases. This led to the formation of persistent dendrites and branches. Additional timelapse studies on the dynamics of dendrite elongation showed alternating periods of elongation and retraction. Phosphatidylinositol 3-kinases increased the velocities of dendrite elongation and retraction, whereas the neuropeptides prolonged the periods of elongation. By modifying NMDA-induced activation of Rho GTPases and phosphatidylinositol 3-kinases, vasoactive intestinal peptide and PACAP38 could play an important role in the control of dendrite growth and branching during development and in response to neuronal activity.

Toxic Effects of Lipid‐Mediated Gene Transfer in Ventral Mesencephalic Explant Cultures

Adverse effects of cDNA and oligonucleotide delivery methods have not yet been systematically analyzed. We introduce a protocol to monitor toxic effects of two non-viral lipid-based gene delivery protocols using CNS primary tissue. Cell membrane damage was monitored by quantifying cellular uptake of propidium iodide and release of cytosolic lactate dehydrogenase to the culture medium. Using a liposomal transfection reagent, cell membrane damage was already seen 24 hr after transfection. Nestin-positive target cells, which were used as morphological correlate, were severely diminished in some areas of the cultures after liposomal transfection. In contrast, the non-liposomal transfection reagent revealed no signs of toxicity. This approach provides easily accessible information of transfection-associated toxicity and appears suitable for prescreening of transfection reagents.

Ionotropic glutamate receptors and glutamate transporters are involved in necrotic neuronal cell death induced by oxygen–glucose deprivation of hippocampal slice cultures

Organotypic hippocampal slice cultures represent a feasible model for studies of cerebral ischemia and the role of ionotropic glutamate receptors in oxygen–glucose deprivation-induced neurodegeneration. New results and a review of existing data are presented in the first part of this paper. The role of glutamate transporters, with special reference to recent results on inhibition of glutamate transporters under normal and energy-failure (ischemia-like) conditions is reviewed in the last part of the paper. The experimental work is based on hippocampal slice cultures derived from 7 day old rats and grown for about 3 weeks. In such cultures we investigated the subfield neuronal susceptibility to oxygen–glucose deprivation, the type of induced cell death and the involvement of ionotropic glutamate receptors. Hippocampal slice cultures were also used in our studies on glutamate transporters reviewed in the last part of this paper. Neurodegeneration was monitored and/or shown by cellular uptake of propidium iodide, loss of immunocytochemical staining for microtubule-associated protein 2 and staining with Fluoro-Jade B. To distinguish between necrotic vs. apoptotic neuronal cell death we used immunocytochemical staining for active caspase-3 (apoptosis indicator) and Hoechst 33342 staining of nuclear chromatin. Our experimental studies on oxygen–glucose deprivation confirmed that CA1 pyramidal cells were the most susceptible to this ischemia-like condition. Judged by propidium iodide uptake, a selective CA1 lesion, with only minor affection on CA3, occurred in cultures exposed to oxygen–glucose deprivation for 30 min. Nuclear chromatin staining by Hoechst 33342 and staining for active caspase-3 showed that oxygen–glucose deprivation induced necrotic cell death only. Addition of 10μM of the N-methyl- d-aspartate glutamate receptor antagonist MK-801, and 20μM of the non- N-methyl- d-aspartate glutamate receptor antagonist 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo( F)quinoxaline to the culture medium confirmed that both N-methyl- d-aspartate and non- N-methyl- d-aspartate ionotropic glutamate receptors were involved in the oxygen–glucose deprivation-induced cell death. Glutamate is normally quickly removed, from the extracellular space by sodium-dependent glutamate transporters. Effects of blocking the transporters by addition of the dl-threo-beta-benzyloxyaspartate are reviewed in the last part of the paper. Under normal conditions addition of dl-threo-beta-benzyloxyaspartate in concentrations of 25μM or more to otherwise untreated hippocampal slice cultures induced neuronal cell death, which was prevented by addition of 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo( F)quinoxaline and MK-801. In energy failure situations, like cerebral ischemia and oxygen–glucose deprivation, the transporters are believed to reverse and release glutamate to the extracellular space. Blockade of the transporters by a subtoxic (10μM) dose of dl-threo-beta-benzyloxyaspartate during oxygen–glucose deprivation (but not during the next 48 h after oxygen–glucose deprivation) significantly reduced the oxygen–glucose deprivation-induced propidium iodide uptake, suggesting a neuroprotective inhibition of reverse transporter activity by dl-threo-beta-benzyloxyaspartate during oxygen–glucose deprivation under these conditions. Adding to this, other results from our laboratory have demonstrated that pre-treatment of the slice cultures with glial cell-line derived neurotrophic factor upregulates glutamate transporters. As a logical, but in some glial cell-line derived neurotrophic factor therapy-related conditions clearly unwanted consequence the susceptibility for oxygen–glucose deprivation-induced glutamate receptor-mediated cell death is increased after glial cell-line derived neurotrophic factor treatment. In summary, we conclude that both ionotropic glutamate receptors and glutamate transporters are involved in oxygen–glucose deprivation-induced necrotic cell death in hippocampal slice cultures, which have proven to be a feasible tool in experimental studies on this topic.

Tetraethylammonium exacerbates ischemic neuronal injury in rat cerebrocortical slice cultures

We investigated potential contribution of K + channel activity to regulation of ischemia-induced neuronal injury, using cerebrocortical slice cultures. Exposure of cultures to a glucose-free conditioning solution containing sodium azide and 2-deoxyglucose caused neuronal cell death as assessed by cellular uptake of propidium iodide, which was prevented by MK-801, an N-methyl- d-aspartate (NMDA) receptor antagonist. Application of tetraethylammonium markedly exacerbated ischemic neuronal injury. Charybdotoxin, a blocker of large-conductance Ca 2+-activated K + (BK Ca) channels, also augmented ischemic injury, whereas AM 92016, a blocker of delayed rectifier K + channels, and dequalinium, a blocker of small-conductance Ca 2+-activated K + channels, had no significant effect. In addition, tetraethylammonium and charybdotoxin were effective in augmenting NMDA-induced neuronal injury. These results present unprecedented evidence for the ability of tetraethylammonium to enhance ischemic neuronal death, and suggest that BK Ca channels constitute an endogenous system to protect cortical neurons from ischemic injury, via prevention of NMDA receptor over-activation.

Neuroprotection against NMDA excitotoxicity by group I metabotropic glutamate receptors is associated with reduction of NMDA stimulated currents

The neurotransmitter glutamate can have both excitotoxic and protective effects on neurons. The excitotoxic effects have been intensively studied, whereas the protective effects, including the involvement of metabotropic glutamate receptors (mGluRs), remain unclear. In the present study, we tested the protective effects of the group-I-mGluR agonist ( S)-3,5-dihydroxyphenylglycine (DHPG) on organotypic hippocampal slice cultures exposed to excitotoxic concentrations of N-methyl- d-aspartate (NMDA). Effects of DHPG on electrophysiological responses induced by NMDA receptor activation were also recorded. Experiments were performed on organotypic hippocampal slice cultures derived from 7-day-old rats, with cellular uptake of propidium iodide as a marker for neuronal cell death. Slice cultures pretreated with DHPG (10 or 100 μM) for 2 h prior to exposure to 50 μM NMDA for 30 min displayed reduced propidium iodide uptake, compared to cultures exposed to NMDA only. The neuroprotective effect was confirmed by Hoechst 33342 staining, where the appearance of pycnotic nuclei after NMDA treatment was prevented by the DHPG pretreatment. Using caspase-3 activity to monitor the presence of apoptosis, failed to demonstrate this type of cell death in CA1 after NMDA application. The protective effect of DHPG was abolished by the mGluR1 selective antagonist ( S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385; 5 or 10 μM), whereas the mGluR5-selective antagonist 2-methyl-6-phenylethynylpyridine (MPEP; 1 μM) had no effect. Voltage-clamping of CA1 pyramidal cells in cultures treated with 10 μM DHPG for 2 h showed a significant depression of NMDA-induced inward currents compared to untreated controls. We conclude that neuroprotection induced by activation of group-I-mGluRs involve mGluR1 and is associated with decreased NMDA-stimulated currents.

Tetrahydrobiopterin precursor sepiapterin provides protection against neurotoxicity of 1-methyl-4-phenylpyridinium in nigral slice cultures.

Complex-I inhibition and oxidative processes have been implicated in the loss of nigral dopamine neurones in Parkinson's disease and the toxicity of MPTP and its metabolite MPP+. Tetrahydrobiopterin, an essential cofactor for tyrosine hydroxylase, may act as an antioxidant in dopaminergic neurones and protects against the toxic consequences of glutathione depletion. Here we studied the effects of manipulating tetrahydrobiopterin levels on MPP+ toxicity in organotypic, rat ventral mesencephalic slice cultures. In cultures exposed to 30 micro m MPP+ for 2 days, followed by 8 days 'recovery' in control medium, we measured dopamine and its metabolites in the tissue and culture medium by HPLC, lactate dehydrogenase release to the culture medium, cellular uptake of propidium iodide and counted the tyrosine hydroxylase-immunoreactive neurones. Inhibition of tetrahydrobiopterin synthesis by 2,4-diamino-6-hydroxypyrimidine had no significant synergistic effect on MPP+ toxicity. In contrast, the tetrahydrobiopterin precursor l-sepiapterin attenuated the MPP+-induced dopamine depletion and loss of tyrosine hydroxylase-positive cells in a dose-dependent manner with 40 micro m l-sepiapterin providing maximal protection. Accordingly, increasing intracellular tetrahydrobiopterin levels may protect against oxidative stress by complex-I inhibition.

Neurotoxic and neuroprotective effects of the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate (dl-TBOA) during physiological and ischemia-like conditions

Maintenance of low extracellular glutamate ([Glu]O) preventing excitotoxic cell death requires fast removal of glutamate from the synaptic cleft. This clearance is mainly provided by high affinity sodium-dependent glutamate transporters. These transporters can, however, also be reversed and release glutamate to the extracellular space in situations with energy failure. In this study the cellular localisation of the glutamate transporters GLAST and GLT-1 in organotypic hippocampal slice cultures was studied by immunofluorescence confocal microscopy, under normal culture conditions, and after a simulated ischemic insult, achieved by oxygen and glucose deprivation (OGD). In accordance with in vivo findings, GLAST and GLT-1 were primarily expressed by astrocytes under normal culture conditions, but after OGD some damaged neurons also expressed GLAST and GLT-1. The potential damaging effect of inhibition of the glutamate transporters by dl-threo-beta-benzyloxyaspartate (dl-TBOA) was studied using cellular uptake of propidium iodide (PI) as a quantitative marker for the cell death. Addition of dl-TBOA for 48h was found to induce significant cell death in all hippocampal regions, with EC50 values ranging from 38 to 48μM for the different hippocampal subregions. The cell death was prevented by addition of the glutamate receptor antagonists NBQX and MK-801, together with an otherwise saturating concentration of dl-TBOA (100μM). Finally, the effect of inhibition of glutamate release, via reverse operating transporters during OGD, was investigated. Addition of a sub-toxic (10μM) dose of dl-TBOA during OGD, but not during the subsequent 48h recovery period, significantly reduced the OGD-induced PI uptake. It is concluded: (1) that the cellular expression of the glutamate transporters GLAST and GLT-1 in hippocampal slice cultures in general corresponds to the expression in vivo, (2) that inhibition of the glutamate transporters induces cell death in the slice cultures, and (3) that partial inhibition during simulation of ischemia by OGD protects against the induced PI uptake, most likely by blocking the reverse operating transporters otherwise triggered by the energy failure.

Domoic acid neurotoxicity in hippocampal slice cultures.

The neurotoxicity of domoic acid was studied in 2-3 week old rat hippocampal slice cultures, derived from 7 day old rat pups. Domoic acid 0.1-100 microM was added to the culture medium for 48 hrs, alone or together with the glutamate receptor antagonists NS-102 (5-Nitro-6,7,8,9-tetrahydrobenzo[G]indole-2,3-dione-3-oxime), NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline) or MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), followed by transfer of the cultures to normal medium for additional 48 hrs. Neuronal degeneration in the fascia dentata (FD), CA3 and CA1 hippocampal subfields was monitored and EC(50) values estimated by densitometric measurements of the cellular uptake of propidium iodide (PI). The CA1 region was most sensitive to domoic acid, with an EC(50) value of 6 microM domoic acid, estimated from the PI-uptake at 72 hrs. Protective effects of 10 microM NBQX against 3 and 10 microM domoic acid were observed for both dentate granule cells and CA1 and CA3c pyramidal cells. NS102 and MK 801 only displayed protective effects when combined with NBQX. MK801 significantly increased the combined neuroprotective effect of NBQX and NS102 against 10 microM domoic acid in both CA1 and FD, but not in CA3. We conclude, that domoic acid neurotoxicity in CA3 and in hippocampal slice cultures in general primarily involves AMPA/kainate receptors. At high concentrations (10 microM domic acid) NMDA receptors are, however, also involved in the toxicity in CA1 and FD.

Pilocarpine-induced seizure-like activity with increased BNDF and neuropeptide Y expression in organotypic hippocampal slice cultures

Organotypic hippocampal slice cultures were treated with the muscarinic agonist pilocarpine to study induced seizure-like activity and changes in neurotrophin and neuropeptide expression. For establishment of a seizure-inducing protocol, 2-week-old cultures derived from 6–8-day-old rats were exposed to 0.1 mM to 5 mM of pilocarpine for 4 h to 7 days. Other cultures were treated with pilocarpine for 7 days and left for 7–14 days in normal medium. Age-matched, non-treated cultures served as controls. Intracellular recordings from CA1 pyramidal cells revealed increased spontaneous activity in 31 of 35 cultures superfused with 0.1 or 5 mM pilocarpine. Epileptiform discharges were recorded in 17 of the 31 cultures, and 19 displayed frequencies specifically in the 6–12-Hz (Theta rhythm) range when superfused with pilocarpine. The pilocarpine effect was blocked by simultaneous superfusion with the muscarinic receptor antagonist atropine (100 μM). Regardless of dose and exposure time, the pilocarpine treatment induced very limited neuronal cell death, recorded as cellular propidium iodide uptake. Cultures exposed to 5 mM pilocarpine for up to 7 days displayed increased BDNF expression when analyzed by Western blot and ELISA. This BDNF increase correlated with increased neuropeptide Y immunoreactivity, known to accompany seizure activity. Addition of BDNF (200 ng/ml) to otherwise untreated cultures also upregulated NPY expression. The pilocarpine-induced seizure-like activity in hippocampal slice cultures, with concomitant increase in BDNF and NPY expression, is compared with in vivo observations and discussed in terms of the potential use of the easily accessible slice cultures in experimental seizure research.

Nuclear shrinkage and other markers of neuronal cell death after oxygen–glucose deprivation in rat hippocampal slice cultures

Organotypic hippocampal slice cultures are used increasingly in experimental models of neurodegeneration, together with various histological, biochemical and electrophysiological markers of cell death. While functional electrophysiological changes typically occur early, histological changes appear later, with loss of dendritic immunoreactivity for microtubule-associated protein 2 (MAP2) among the earliest. In this study we compared the temporal changes of four different histological markers for neurodegeneration after oxygen–glucose deprivation (OGD) of rat hippocampal slice cultures. Within an observation period of 24 h after OGD, shrinkage of Hoechst 33342 stained neuronal nuclei both occurred before, and was completed faster, than loss of MAP2 staining, which again started earlier and progressed faster towards complete loss than the increase in cellular uptake of propidium iodide and Fluoro-Jade B staining of degenerating neurons. We conclude that shrinkage of Hoechst 33342 stained neuronal nuclei detected by image analysis is an early and easily quantifiable indicator of neuronal degeneration in hippocampal slice cultures.

Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures

The excitotoxic profiles of ( RS)-2-amino-3-(3-hydroxy-5- tert-butylisoxazol-4-yl)propionic acid (ATPA), ( RS)-2-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainic acid (KA) and N-methyl- d-aspartate (NMDA) were evaluated using cellular uptake of propidium iodide (PI) as a measure for induced, concentration-dependent neuronal damage in hippocampal slice cultures. ATPA is in low concentrations a new selective agonist of the glutamate receptor subunit GluR5 confined to KA receptors and also in high concentrations an AMPA receptor agonist. The following rank order of estimated EC 50 values was found after 2 days of exposure: AMPA (3.7 mM)>NMDA (11 mM)=KA (13 mM)>ATPA (33 mM). Exposed to 30 μM ATPA, 3 μM AMPA and 10 μM NMDA, CA1 was the most susceptible subfield followed by fascia dentata and CA3. Using 8 μM KA, CA3 was the most susceptible subfield, followed by fascia dentata and CA1. In 100 μM concentrations, all four agonists induced the same, maximal PI uptake in all hippocampal subfields, corresponding to total neuronal degeneration. Using glutamate receptor antagonists, like GYKI 52466, NBQX and MK-801, inhibition data revealed that AMPA excitotoxicity was mediated primarily via AMPA receptors. Similar results were found for a high concentration of ATPA (30 μM). In low GluR5 selective concentrations (0.3–3 μM), ATPA did not induce an increase in PI uptake or a reduction in glutamic acid decarboxylase (GAD) activity of hippocampal interneurons. For KA, the excitotoxicity appeared to be mediated via both KA and AMPA receptors. NMDA receptors were not involved in AMPA-, ATPA- and KA-induced excitotoxicity, nor did NMDA-induced excitotoxicity require activation of AMPA and KA receptors. We conclude that hippocampal slice cultures constitute a feasible test system for evaluation of excitotoxic effects and mechanisms of new (ATPA) and classic (AMPA, KA and NMDA) glutamate receptor agonists. Comparison of concentration–response curves with calculation of EC 50 values for glutamate receptor agonists are possible, as well as comparison of inhibition data for glutamate receptor antagonists. The observation that the slice cultures respond with more in vivo-like patterns of excitotoxicity than primary neuronal cultures, suggests that slice cultures are the best model of choice for a number of glutamate agonist and antagonist studies.

The metabotropic glutamate receptor agonist 1 S,3 R-ACPD stimulates and modulates NMDA receptor mediated excitotoxicity in organotypic hippocampal slice cultures

The potential toxic effects of the metabotropic glutamate receptor agonist (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) and its interactions with the N-methyl- d-aspartate (NMDA) receptor were studied in hippocampal brain slice cultures, using densitometric measurements of the cellular uptake of propidium iodide (PI) to quantify neuronal degeneration. Cultures exposed to ACPD, showed a concentration (2–5 mM) and time (1–4 days) dependent increase in PI uptake in CA1, CA3 and dentate subfields after 24 h and 48 h of exposure, with CA1 pyramidal cells being most sensitive. The neurodegeneration induced by 2 mM ACPD was completely abolished by addition of 10 μM of the NMDA receptor antagonist (5 R,10 S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[ a, d]cyclohepten-5,10-imine (MK-801), while 20 μM of the 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainic acid receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[ f]quinoxaline-7-sulfonamide (NBQX) had no effect. Co-exposing cultures to a subtoxic dose of 300 μM ACPD together with 10 μM NMDA, which at this dose is known to induce a fairly selective degeneration of CA1 pyramidal cells, significantly increased the PI uptake in both CA1 and CA3, compared to cultures exposed to 10 μM NMDA only. Adding the 300 μM ACPD as pretreatment for 30 min followed by a 30 min wash in normal medium before the ACPD/NMDA co-exposure, eliminated the potentiation of NMDA toxicity. The potentiation was also blocked by addition of 10 or 100 μM 2-methyl-6-(phenylethynyl)pyridine (MPEP) (mGluR5 antagonist) during the co-exposure, while a corresponding addition of 10 or 100 μM 7-(hydroxyimino)cyclopropa[ b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (mGluR1 antagonist) had no effect. We conclude that, stimulation of metabotropic glutamate receptors with ACPD at concentrations of 2 mM or higher induces a distinct subfield-related and time and concentration dependent pattern of hippocampal degeneration, and that ACPD at subtoxic concentrations modulates NMDA-induced excitotoxicity through the mGluR5 receptor in a time dependent way.